Functional Characterization of the Wheat Macrophage Migration Inhibitory Factor TaMIF1 in Wheat-Stripe Rust (Puccinia striiformis) Interaction

Simple Summary There have been many breakthroughs in MIF function and mechanism investigation in vertebrates, but it has rarely been studied in plants. Here, we aimed to characterize the function of MIF in wheat and its potential role in Wheat-Stripe rust interaction. We showed that wheat MIF has some similarities with that MIF in vertebrates, such as subcellular localization in both the cytosol and nuclei, as well as significant tautomerase activity, and both can inhibit Bax-induced programmed cell death. In the wheat–Pst interaction, TaMIF1 is upregulated during Pst infection. Silencing TaMIF1 decreased Pst infection of wheat tissues, and the accumulation of ROS was increased in TaMIF1-silenced wheat leaves, which hinted that TaMIF1 mainly modulates the ROS signaling and then alters the subsequent immune responses. The function characterization of TaMIF1 provides significant insight into the role of MIFs across kingdoms and helpful in-depth functional mechanism analysis on these proteins. Abstract Macrophage migration inhibitory factor (MIF), named for its role in inhibiting macrophage/monocyte migration, has multiple functions in modulation of inflammation, cell proliferation, angiogenesis, and tumorigenesis in vertebrates. Although homologs of this gene can be found in plants, the function of MIF in plants remains obscure. Here, we characterized TaMIF1 in Triticum aestivum resembling the MIF secreted from Homo sapiens. Transcript analysis revealed that TaMIF1 responded to stripe rust infection of wheat and was upregulated during the infection stage. TaMIF1 was localized to both the cytosol and nuclei in wheat mesophyll protoplast. Additionally, TaMIF1 possessed significant tautomerase activity, indicating conservation of MIFs across kingdoms. Agrobacterium tumefaciens infiltration assay demonstrated that TaMIF1 was capable of suppressing programmed cell death hinting its role in plant immunity. Heterologous expression of TaMIF1 increased fission yeast sensitivity to oxidative stress. Silencing TaMIF1 decreased the susceptibility of wheat to Pst seemingly through increasing reactive oxygen species accumulation. In conclusion, functions of the TaMIF1 were investigated in this study, which provides significant insight into understanding the role of MIFs across kingdoms.


Introduction
Macrophage migration inhibitory factor (MIF) is firstly identified from the culture supernatant of activated T lymphocytes, with the role of inhibiting the migration of macrophages [1]. In vertebrates, MIF is not only expressed within the cytosol of immune cells, but also expressed in various non-immune cells during the regulation of cellular two-leaf stage. CYR31 and CYR23 were grown and collected from wheat cultivar Suwon11 and Mingxian169, respectively [27]. Escherichia coli strain BL21 was used for prokaryotic expression system. Fission yeast isolate Schizosaccharomyces pombe and A. tumefaciens strain GV3101 were used for gene expression.

Plasmid Constructs
Primers used throughout are found in the Supplementary Information (Table S1). For the subcellular localization of TaMIF1 assay, ORF of TaMIF1 was cloned into the vector pTF486 [28]. PVX vector pGR107 was used to heterogeneously overexpress TaMIF1 in N. benthamiana [29]. Vector pREP3X was used for overexpression of TaMIF1 in fission yeast cells [30]. TaMIF1 was constructed in vector pGEX-4T-1 for prokaryotic expression. The BSMV vector was used for TaMIF1 silencing [31].

Sequence Analysis and Domain Prediction
EST sequence of TaMIF1 was from the cDNA library of wheat-Pst [26]. The obtained cDNA was cloned and aligned with the wheat genome database at EnsemblPlants (http://plants.ensembl.org/index.html accessed on 18 January 2021). All nucleotide and amino acid sequences were aligned using DNAMAN6. The expression profiles of TaMIF genes in different development stages were determined on the basis of the RNAseq data available from WheatOmics 1.0 (http://202.194.139.32/ accessed on 8 May 2021). The amino acid sequence of MIF from H. sapiens (accession no. NP_002406) was obtained from GenBank (http://www.ncbi.nlm.nih.gov accessed on 12 December 2020). The amino acid sequences of the three MIFs from A. thaliana were obtained from TAIR (http://www.arabidopsis.org/ accessed on 12 December 2020). Characteristic MIF domains were predicted with InterProScan 5 (http://www.ebi.ac.uk/interpro/ accessed on 12 December 2020). Nuclear export signal was predicted in NetNES 1.1 (http://www.cbs.dtu. dk/services/NetNES/ accessed on 12 December 2020).

RNA Extraction and qRT-PCR
Total RNAs were extracted using an RNAeasy R Plant Mini Kit (Qiagen, Shenzhen, China). The first-strand cDNA was synthesized using the Go Script Reverse Transcription System (Promega, Madison, WI, USA). Transcript profiles were measured using qRT-PCR. TaEF-1 (T. aestivum elongation factor gene with accession no. Q03033) was used for normalization. A CFX Connect real-time PCR system (Bio-Rad, Singapore) was used to perform qRT-PCR, and the transcript levels were calculated using the 2 −∆∆CT method [32].

Subcellular Localization of TaMIF1 in Wheat Protoplast
The wheat protoplast preparation refers to the protocol used for A. thaliana [33]. The plasmids carrying TaMIF1-GFP was transformed into wheat protoplast and incubated for 18 h in the dark. The fluorescence of transformed protoplasts was checked using the fluorescence microscope (Olympus Corporation, Tokyo, Japan) with the 485 nm laser line. Total protein was isolated from wheat protoplasts as previously described [34]. Western blotting was used for verifying protein expression. Detecting antibodies of mouse-derived GFP antibody and horseradish peroxidase-conjugated anti-mouse secondary antibody were all from commercial purchase (Sungene, Tianjin, China). The chemiluminescence substrate Pierce™ ECL Western Blotting Substrate was used for detection (Thermo Fisher Scientific, Waltham, MA, USA).

Heterogeneous Agroinfiltration Assays
Strain GV3101 carrying the expression plasmids were cultured to the late log phase. The infiltration buffer was used to resuspend cells to an OD600 of 0.2. Before infiltration, cells needed to be incubated in the dark for 2 h. Cell suspension carrying TaMIF1 and Bax were infiltrated into the tobacco leaves one day apart. Photos were taken after 3-5 days' infiltration. In order to degrade chlorophyll, we decolorized the leaves using ethanol. eGFP was used as the negative control. The positive control was the oomycete elicitor Avr1b that suppressed Bax-induced PCD [35]. Western blotting was used to detect protein expression.

Tautomerase Activity Assay
pGEX-4T-TaMIF1 was expressed in E. coli strain BL21. Proteins were induced and collected after incubating at LB medium with 1.0 mM IPTG overnight at 16 • C. Cells were collected and lysed by sonication. After centrifugation, soluble proteins were in the supernatant and analyzed by SDS-PAGE. For further tautomerase activity assay, GSTrap was used to purify the fusion proteins. Tautomerase activity was measured using Ldopachrome methyl ester [36,37], which was prepared just before use by adding L-3,4dihydroxuphenylalanine methyl ester (4 mM) and sodium periodate (8 mM) to a reaction buffer (1 mM EDTA, pH 6.2). The mixture was incubated for 5 min at room temperature and placed on ice for 20 min. The activity was determined at room temperature by adding 800 µL of the prepared L-dopachrome methyl ester to a cuvette containing TaMIF1 or MmMIF expressed from a prokaryotic expression system and measuring the decrease in absorbance at 475 nm for 15 min. The rate decrease of absorbance was calculated after 60 s. Empty vector pGEX-4T was used as negative control and MmMIF was used as the positive control.

The Heterogenous Overexpression in Fission Yeast Assay
pREP3X-TaMIF1 was conducted and transformed into S. pombe by electroporation. Thiamine was the repressor that can inhibit promoter of pREP3X. Transformed cells diluted to OD600 = 0.2 were cultured in inducing (−Thiamine) or repressing (+Thiamine) medium for about 20 h at 30 • C in the dark. Then, cells were collected by centrifugation; washed with ddH 2 O; and finally diluted to densities of 10 6 , 10 5 , 10 4 , and 10 3 cell mL −1 and removed on yeast medium (adding 0.5 mM H 2 O 2 for H 2 O 2 sensitivity assay and adding 0.35 M NaCl for hyperosmotic stress assay). The morphology of yeast cells was observed using the microscope in brightfield.

Tranient Gene Silencing of TaMIF1 in Wheat cv. Suwon11
Virus-induced gene silencing was carried out as described [31,38]. BSMV:TaMIF-1as and BSMV:TaMIF-2as were constructed and used for inoculating the second leaves of wheat seedlings. BSMV:TaPDS-as and BSMV:00 were used as controls. Ten days after viral inoculation, the fourth leaves of wheat seedlings were inoculated with stripe rust of fresh CYR31 urediospores. Phenotypes of the fourth leaves were observed and photographed 14 days after Pst inoculation. Pst-challenged fourth leaves of wheat seedlings inoculated with BSMV in advance were collected for RNA extraction at 0, 12, 24, 48, and 120 hpi. qRT-PCR was used to evaluate the silencing efficiencies of TaMIF1 using the RNA extracted at each time point.

Histological Sample Treatment and Observation
DAB staining was used to observe H 2 O 2 accumulation [39]. Virus-inoculated leaves were infected with CYR31 and sampled at 12, 24, and 48 hpi. Leaf segments were treated in ethanol/acetic acid (1:1, v/v). Samples were immersed in DAB solution for 8 h under light at room temperature. Infection structures of Pst were stained with wheat germ agglutinin conjugated to Alexa 488 (WGA488) (Invitrogen, Carlsbad, CA, USA) [40]. Virus-inoculated leaves were sampled at 24 and 120 hpi with CYR31 infection. The decolorized samples were autoclaved in 1M KOH for 2 min, washed twice with 50 mM Tris (pH 7.5), and stained with WGA488 (20 µg/mL) for 30 min. The stained samples were viewed with the fluorescence microscope and measured using DP-BSW software (Olympus Corporation).

Genome-Wide Identification of MIF-Like Genes in Wheat and Their Expression
On the basis of the EST sequence WRIS 3105 from the cDNA library of Pst-wheat interaction [26], a MIF-like gene with an ORF of 348 bp was identified and cloned from the wheat cultivar Suwon11. Sequence analysis revealed that the isolated wheat MIF gene (designated as TaMIF1) has three copies located on wheat chromosomes 5A, 5B, and 5D. Another paraologuous MIF gene (designated as TaMIF2) was also searched in the wheat genome, being found to be located on chromosomes 7A, 7B, and 7D. The nucleotide sequences of the two group genes were found to be highly similar (82.85%), making it difficult to study individual TaMIF expression profiles. However, according to transcription analysis, homologous genes on chromosomes 5D and 5B responded to the induction of Pst, and homologs on 5A, 7A, 7B, and 7D were not induced ( Figure 1A). Thus, we chose TaMIF1 as a representative to carry out further research. In particular, we found that TaMIF1 has two transcripts. To identify expression profiles of different transcripts in wheat and Pst interaction, we designed specific primers for qRT-PCR ( Figure S1). The results in Figure 1B indicate that the two transcripts have similar expression profiles. The transcript levels are both induced during Pst infection ( Figure 1B). the fluorescence microscope and measured using DP-BSW software (Olympus Corporation).

Genome-Wide Identification of MIF-Like Genes in Wheat and Their Expression
On the basis of the EST sequence WRIS 3105 from the cDNA library of Pst-wheat interaction [26], a MIF-like gene with an ORF of 348 bp was identified and cloned from the wheat cultivar Suwon11. Sequence analysis revealed that the isolated wheat MIF gene (designated as TaMIF1) has three copies located on wheat chromosomes 5A, 5B, and 5D. Another paraologuous MIF gene (designated as TaMIF2) was also searched in the wheat genome, being found to be located on chromosomes 7A, 7B, and 7D. The nucleotide sequences of the two group genes were found to be highly similar (82.85%), making it difficult to study individual TaMIF expression profiles. However, according to transcription analysis, homologous genes on chromosomes 5D and 5B responded to the induction of Pst, and homologs on 5A, 7A, 7B, and 7D were not induced ( Figure 1A). Thus, we chose TaMIF1 as a representative to carry out further research. In particular, we found that TaMIF1 has two transcripts. To identify expression profiles of different transcripts in wheat and Pst interaction, we designed specific primers for qRT-PCR ( Figure S1). The results in Figure 1B    Through sequence alignment and domain prediction, we found that wheat MIF proteins have similar domains to HsMIF and MIFs in A. thaliana. Amino acid sequence alignment of HsMIF, AtMIFs, and TaMIFs indicates that they all contain three conserved residues (Pro1, Lys32, and Ile64) of the active sites for tautomerase activity identified in HsMIF ( Figure 1C, black triangle), but plant MIFs lack the C-X-X-C motif that conducts the oxidoreductase activity. Our results suggest that TaMIFs are phylogenetically ancient proteins that seem evolutionarily conserved.

TaMIF1 Showed Tautomerase Activity
With the prediction by ExPASy, a tautomerase domain was found in TaMIF1 as all MIFs reported. Thus, the protein of TaMIF1 was expressed in a prokaryotic expression system to investigate its enzymatic activity ( Figure S2). In this study, MmMIF, the MIF from Mus musculus, was used as a positive control. As shown by the results, with L-dopachrome methyl ester as the substrate, TaMIF1 and MmMIF displayed significant tautomerase activity as assessed by the decreasing absorbance at 475 nm ( Figure 2). This result suggests conservation between TaMIF1 and MIFs from vertebrates in the tautomerase activity. Through sequence alignment and domain prediction, we found that wheat MIF proteins have similar domains to HsMIF and MIFs in A. thaliana. Amino acid sequence alignment of HsMIF, AtMIFs, and TaMIFs indicates that they all contain three conserved residues (Pro1, Lys32, and Ile64) of the active sites for tautomerase activity identified in HsMIF ( Figure 1C, black triangle), but plant MIFs lack the C-X-X-C motif that conducts the oxidoreductase activity. Our results suggest that TaMIFs are phylogenetically ancient proteins that seem evolutionarily conserved.

TaMIF1 Showed Tautomerase Activity
With the prediction by ExPASy, a tautomerase domain was found in TaMIF1 as all MIFs reported. Thus, the protein of TaMIF1 was expressed in a prokaryotic expression system to investigate its enzymatic activity ( Figure S2). In this study, MmMIF, the MIF from Mus musculus, was used as a positive control. As shown by the results, with L-dopachrome methyl ester as the substrate, TaMIF1 and MmMIF displayed significant tautomerase activity as assessed by the decreasing absorbance at 475 nm ( Figure 2). This result suggests conservation between TaMIF1 and MIFs from vertebrates in the tautomerase activity.

TaMIF1 Was Localized to the Cytosol and the Nuclei
In vertebrates, most MIFs are predominantly stored in cytosols [5], and they could also be detected in small vesicles [41] and the nucleus of vertebrate cells [42]. To identify the subcellular localization of TaMIF1 in wheat, we performed a transient expression of TaMIF1-GFP fusion protein in wheat mesophyll protoplasts. Fluorescence of the fusion protein was observed in both the cytoplasm and the nucleus, as was fluorescence of the control expressing GFP coding sequence only ( Figure 3). Expression of GFP and the TaMIF1-GFP fusion were confirmed by Western blot analysis ( Figure S3). These results indicate that TaMIF1 localizes to both the cytoplasm and nucleus in wheat cells.

TaMIF1 Was Localized to the Cytosol and the Nuclei
In vertebrates, most MIFs are predominantly stored in cytosols [5], and they could also be detected in small vesicles [41] and the nucleus of vertebrate cells [42]. To identify the subcellular localization of TaMIF1 in wheat, we performed a transient expression of TaMIF1-GFP fusion protein in wheat mesophyll protoplasts. Fluorescence of the fusion protein was observed in both the cytoplasm and the nucleus, as was fluorescence of the control expressing GFP coding sequence only ( Figure 3). Expression of GFP and the TaMIF1-GFP fusion were confirmed by Western blot analysis ( Figure S3). These results indicate that TaMIF1 localizes to both the cytoplasm and nucleus in wheat cells. Biology 2021, 10, x FOR PEER REVIEW 7 of 15

Transient Expression of TaMIF1 in Tobacco Suppressed Programmed Cell Death
In vertebrates, MIFs are important regulators of cell apoptosis, which sustains macrophage survival and suppresses p53-dependent apoptosis in vertebrates [17,43]. It is interesting to note that the BCL2-associated X (Bax) protein from mammals can trigger programmed cell death (PCD) when expressed in plants from a tobacco mosaic virus vector (PVX) [44]. To determine whether TaMIF1 is related to PCD, we overexpressed TaMIF1 in N. benthamiana using the PVX vector. When leaves were infiltrated with eGFP (negative control), Avr1b (positive control) [35], or TaMIF1, no cell death was observed. The site inoculated with eGFP then challenged by Agrobacterium expressing Bax 24 h later exhibited obvious cell death, while the sites inoculated with Agrobacterium expressing Avr1b or TaMIF1 then challenged with Bax showed no cell death, indicating that the cell death induced by Bax was successfully suppressed by Avr1b or TaMIF1 (Figure 4). Western blotting of proteins expression are shown in Figure S4. The result indicates that TaMIF1 suppresses PCD directly in plants.

Transient Expression of TaMIF1 in Tobacco Suppressed Programmed Cell Death
In vertebrates, MIFs are important regulators of cell apoptosis, which sustains macrophage survival and suppresses p53-dependent apoptosis in vertebrates [17,43]. It is interesting to note that the BCL2-associated X (Bax) protein from mammals can trigger programmed cell death (PCD) when expressed in plants from a tobacco mosaic virus vector (PVX) [44]. To determine whether TaMIF1 is related to PCD, we overexpressed TaMIF1 in N. benthamiana using the PVX vector. When leaves were infiltrated with eGFP (negative control), Avr1b (positive control) [35], or TaMIF1, no cell death was observed. The site inoculated with eGFP then challenged by Agrobacterium expressing Bax 24 h later exhibited obvious cell death, while the sites inoculated with Agrobacterium expressing Avr1b or TaMIF1 then challenged with Bax showed no cell death, indicating that the cell death induced by Bax was successfully suppressed by Avr1b or TaMIF1 (Figure 4). Western blotting of proteins expression are shown in Figure S4. The result indicates that TaMIF1 suppresses PCD directly in plants.

Transient Expression of TaMIF1 in Tobacco Suppressed Programmed Cell Death
In vertebrates, MIFs are important regulators of cell apoptosis, which sustains macrophage survival and suppresses p53-dependent apoptosis in vertebrates [17,43]. It is interesting to note that the BCL2-associated X (Bax) protein from mammals can trigger programmed cell death (PCD) when expressed in plants from a tobacco mosaic virus vector (PVX) [44]. To determine whether TaMIF1 is related to PCD, we overexpressed TaMIF1 in N. benthamiana using the PVX vector. When leaves were infiltrated with eGFP (negative control), Avr1b (positive control) [35], or TaMIF1, no cell death was observed. The site inoculated with eGFP then challenged by Agrobacterium expressing Bax 24 h later exhibited obvious cell death, while the sites inoculated with Agrobacterium expressing Avr1b or TaMIF1 then challenged with Bax showed no cell death, indicating that the cell death induced by Bax was successfully suppressed by Avr1b or TaMIF1 (Figure 4). Western blotting of proteins expression are shown in Figure S4. The result indicates that TaMIF1 suppresses PCD directly in plants.  In vertebrates, MIFs play an important role in cell proliferation. To investigate whether TaMIF1 also has a role in cell growth and sensible to some environmental stimuli, we constructed TaMIF1 to the pREP3X vector [30] and transformed pREP3X-TaMIF1 into S. pombe cells. Yeast cells expressing pREP3X-and pREP3X-TaMIF1 were cultured in inducing (−Thiamine) or repressing (+Thiamine) medium, respectively. Although the growth of yeast did not change significantly, microscopic observation revealed that the morphology of yeast cells transformed with pREP3X-TaMIF1 changed from a long strip to a round shape, showing an aggregated state ( Figure 5A). The cell morphology of all transformed yeast appeared to be normal. The statistical results showed that cell length was significantly shorter compared with the cells grown in the repressing medium ( Figure 5B). Therefore, overexpression of TaMIF1 can affect yeast cell length.

Overexpression of TaMIF1 in the Fission Yeast Affected Cell Morphology and Increased the Sensitivity to H2O2
In vertebrates, MIFs play an important role in cell proliferation. To investigate whether TaMIF1 also has a role in cell growth and sensible to some environmental stimuli, we constructed TaMIF1 to the pREP3X vector [30] and transformed pREP3X-TaMIF1 into S. pombe cells. Yeast cells expressing pREP3X-and pREP3X-TaMIF1 were cultured in inducing (−Thiamine) or repressing (+Thiamine) medium, respectively. Although the growth of yeast did not change significantly, microscopic observation revealed that the morphology of yeast cells transformed with pREP3X-TaMIF1 changed from a long strip to a round shape, showing an aggregated state ( Figure 5A). The cell morphology of all transformed yeast appeared to be normal. The statistical results showed that cell length was significantly shorter compared with the cells grown in the repressing medium ( Figure  5B). Therefore, overexpression of TaMIF1 can affect yeast cell length. We further examined the effects of TaMIF1 on the survival of yeast cells subjected to H2O2 (oxidative stress) and NaCl (hyperosmotic stress). As shown in Figure 5C, the viability was severely reduced in the TaMIF1-transformed yeast cells grown on an inducing medium with H2O2 compared with controls. However, no difference appeared in the viability of yeast cells grown on a medium with NaCl ( Figure 5C). The results indicate yeast cells are more sensible to H2O2 when TaMIF1 exogenously overexpressed in yeast cells, which hints that TaMIF1 may be involved in the regulation of reactive oxygen species.

Silencing TaMIF1 Expression Reduced Wheat Susceptibility to Pst
To characterize the role of TaMIF1 in wheat-Pst interaction, we applied barley stripe mosaic virus (BSMV)-mediated gene silencing [31]. Two different fragments were chosen We further examined the effects of TaMIF1 on the survival of yeast cells subjected to H 2 O 2 (oxidative stress) and NaCl (hyperosmotic stress). As shown in Figure 5C, the viability was severely reduced in the TaMIF1-transformed yeast cells grown on an inducing medium with H 2 O 2 compared with controls. However, no difference appeared in the viability of yeast cells grown on a medium with NaCl ( Figure 5C). The results indicate yeast cells are more sensible to H 2 O 2 when TaMIF1 exogenously overexpressed in yeast cells, which hints that TaMIF1 may be involved in the regulation of reactive oxygen species.

Silencing TaMIF1 Expression Reduced Wheat Susceptibility to Pst
To characterize the role of TaMIF1 in wheat-Pst interaction, we applied barley stripe mosaic virus (BSMV)-mediated gene silencing [31]. Two different fragments were chosen to specifically silence TaMIF1 ( Figure 6A). Then, all the possible targets of the two fragments were predicted with the RNAi off-target prediction tool SI-FI 3.2 in both the genome of wheat and Pst (Table S2). At 10 d after inoculation with BSMV, chlorosis was observed in wheat leaves and obvious photobleaching was observed in plants inoculated with BSMV:TaPDSas (TaPDS, wheat phytoene desaturase) ( Figure 6B), indicating that the RNAi system was effective. BSMV-inoculated wheat plants were then infected with CYR31 to show effects of TaMIF1 in the Pst-wheat interaction. In the Pst-wheat interaction system, both BSMV:TaMIF-1as and BSMV:TaMIF-2as inoculated plants showed delayed and decreased pustules formation ( Figure 6C,D). Transcript levels of TaMIF1 in TaMIF1silenced plants were significantly reduced using either of the two fragments ( Figure 6E).
to specifically silence TaMIF1 ( Figure 6A). Then, all the possible targets of the two fragments were predicted with the RNAi off-target prediction tool SI-FI 3.2 in both the genome of wheat and Pst (Table S1). At 10 d after inoculation with BSMV, chlorosis was observed in wheat leaves and obvious photobleaching was observed in plants inoculated with BSMV:TaPDSas (TaPDS, wheat phytoene desaturase) ( Figure 6B), indicating that the RNAi system was effective. BSMV-inoculated wheat plants were then infected with CYR31 to show effects of TaMIF1 in the Pst-wheat interaction. In the Pst-wheat interaction system, both BSMV:TaMIF-1as and BSMV:TaMIF-2as inoculated plants showed delayed and decreased pustules formation ( Figure 6C,D). Transcript levels of TaMIF1 in TaMIF1-silenced plants were significantly reduced using either of the two fragments (Figure 6E). On the basis of the altered susceptibility phenotype of TaMIF1-silenced wheat plants, we assayed cytological changes in VIGS-silenced plants inoculated with Pst. Histological On the basis of the altered susceptibility phenotype of TaMIF1-silenced wheat plants, we assayed cytological changes in VIGS-silenced plants inoculated with Pst. Histological observations of Pst growth and development in wheat plants are shown in Figure S5 and Figure 7. Statistical analysis shows that the lengths of infective hyphae at 24 hpi and the size of the infected area at 120 hpi decreased significantly in the compatible interaction when TaMIF1 was silenced (Figure 7). Thus, silencing TaMIF1 appears to be beneficial for fungal invasion of wheat in the compatible interaction, and wheat seedlings are less susceptible to Pst with TaMIF1 silenced. observations of Pst growth and development in wheat plants are shown in Figures S5 and  7. Statistical analysis shows that the lengths of infective hyphae at 24 hpi and the size of the infected area at 120 hpi decreased significantly in the compatible interaction when TaMIF1 was silenced (Figure 7). Thus, silencing TaMIF1 appears to be beneficial for fungal invasion of wheat in the compatible interaction, and wheat seedlings are less susceptible to Pst with TaMIF1 silenced.

Accumulation of ROS Was Induced in TaMIF1-Silenced Plants
Since silencing TaMIF1 decreases the susceptibility of wheat to Pst, we assessed the plant immune responses in TaMIF1-silenced plants challenged with Pst. ROS is an important marker of host resistance response to pathogen and is usually generated at the infection unit [38,45]. As shown in Figure 7A, H2O2 accumulation mainly occurred in the guard cells in the early stage of 12 hpi, and the H2O2 amount in TaMIF1-knockdown plants was not affected. Significant changes were observed at 24 and 48 hpi. The accumulation of H2O2 per infection site was significantly induced in TaMIF1-silenced plants, no matter whether in guard cells or in attacked mesophyll cells (Figure 8). These results suggest that silencing TaMIF1 induces ROS accumulation in Pst-infected wheat plants and indicates that TaMIF1 might negatively affect the innate immune system of wheat plants.

Accumulation of ROS Was Induced in TaMIF1-Silenced Plants
Since silencing TaMIF1 decreases the susceptibility of wheat to Pst, we assessed the plant immune responses in TaMIF1-silenced plants challenged with Pst. ROS is an important marker of host resistance response to pathogen and is usually generated at the infection unit [38,45]. As shown in Figure 7A, H 2 O 2 accumulation mainly occurred in the guard cells in the early stage of 12 hpi, and the H 2 O 2 amount in TaMIF1-knockdown plants was not affected. Significant changes were observed at 24 and 48 hpi. The accumulation of H 2 O 2 per infection site was significantly induced in TaMIF1-silenced plants, no matter whether in guard cells or in attacked mesophyll cells (Figure 8). These results suggest that silencing TaMIF1 induces ROS accumulation in Pst-infected wheat plants and indicates that TaMIF1 might negatively affect the innate immune system of wheat plants.

Discussion
As one of the first cytokines described, MIF emerged as a pivotal regulator of innate immune systems in vertebrates [5,8]. MIF-like proteins identified in invertebrate species are almost involved in immune escape from hosts [19][20][21]. As a phylogenetically ancient protein, MIFs were also found in almost plant species [22]. Nevertheless, the function of MIFs in plants remains unknown. In this study, we characterized functions of TaMIF1 gene in wheat. Our results show that TaMIF1 acted as a negative immunity regulator of wheat resistance against Pst, suggesting a conservation function in the innate immune system of animals and plants.
Unlike the single MIF copy found in vertebrates, more than one MIF copies were identified in higher plants, which hinted that different MIF copies may exhibit different functions with different expression patterns [22]. There are two MIF genes in wheat, TaMIF1 with three copies located on chromosomes 5A, 5B, 5D, and TaMIF2 with three copies located on chromosomes 7A, 7B, 7D. According to transcription analysis from the wheat expression database, only MIFs on chromosome 5B and 5D are upregulated during wheat-Pst interaction. Although the similarity level of TaMIFs to HsMIF is only 34.48%, the domains and structure of the proteins seem highly conserved. Combined with the

Discussion
As one of the first cytokines described, MIF emerged as a pivotal regulator of innate immune systems in vertebrates [5,8]. MIF-like proteins identified in invertebrate species are almost involved in immune escape from hosts [19][20][21]. As a phylogenetically ancient protein, MIFs were also found in almost plant species [22]. Nevertheless, the function of MIFs in plants remains unknown. In this study, we characterized functions of TaMIF1 gene in wheat. Our results show that TaMIF1 acted as a negative immunity regulator of wheat resistance against Pst, suggesting a conservation function in the innate immune system of animals and plants.
Unlike the single MIF copy found in vertebrates, more than one MIF copies were identified in higher plants, which hinted that different MIF copies may exhibit different functions with different expression patterns [22]. There are two MIF genes in wheat, TaMIF1 with three copies located on chromosomes 5A, 5B, 5D, and TaMIF2 with three copies located on chromosomes 7A, 7B, 7D. According to transcription analysis from the wheat expression database, only MIFs on chromosome 5B and 5D are upregulated during wheat-Pst interaction. Although the similarity level of TaMIFs to HsMIF is only 34.48%, the domains and structure of the proteins seem highly conserved. Combined with the enzymatic activity as a tautomerase found for TaMIF1, we conclude that MIFs are not only conserved in sequence, but also have an overlap in function across kingdoms. MIFs have been subject to various mimicry mechanisms exploited by viruses and parasites and involved in the pathogenesis of septic shock, arthritis, other inflammatory conditions, and even carcinoma [9,10]. In addition, the plant immune responses could be repressed by aphid-secreted MIFs [20]. The function of plant MIFs in plant immune system seems much more intriguing. Transcription analysis showed that TaMIF1 could respond to Pst infection, which hinted that TaMIF1 participates in wheat-Pst interaction.
In vertebrates, MIFs showed multiple expression locations, being constitutively expressed not only in immune system cells but also in epithelial cells that are in direct contact with the host's natural environment [5]. As for subcellular localization, although most MIFs are predominantly stored in cytosols [5], they could also be detected in small vesicles [41] and the nucleus of vertebrate cells [42]. In this study, the expression of TaMIF1-GFP fusion protein suggested that TaMIF1 localized in both the cytoplasm and the nucleus. The only former localization result of MIFs in A. thaliana also localized in the cytosol and the nucleus [22]. This is following the localization of MIFs in vertebrates. This result also suggested the conservation in the localization of MIFs in both vertebrates and plants. In addition, the localization of TaMIF1 might imply some multiple biological functions that are consistent with those in vertebrates [46].
Bax, a member of the Bcl-2 family, triggers cell death in animals [47], while in vertebrates, MIFs are important regulators of cell apoptosis and can suppress Bax-induced cell death [17,48]. It also has been identified that when expressed in plants, Bax can also activate an endogenous cell death program that is very similar to the programmed cell death of plants caused by pathogen infection [44]. In this study, we showed that TaMIF1 could suppress PCD induced by the Bax gene in N. benthamiana, which indicated that MIFs both in animals and plants share the same function of suppressing cell death. In both plants and animals, PCD fulfills the same role of eliminating the diseased cells [49,50]. Thus, the results also hints that TaMIF1 may have a role in plant immunity to pathogens. In this study, silencing of TaMIF1 enhanced immunity to Pst in TaMIF1-knockdown plants. In response to the virulent Pst CYR31, TaMIF1-knockdown plants were less susceptible with delayed and decreased sporulation. Histological observations revealed silencing of TaMIF1 led to increased H 2 O 2 accumulation at neighboring mesophyll cells and infected sites. The induced TaMIF1 expression at 24 and 48 hpi in compatible interaction may be responsible for the suppression of H 2 O 2 production. The increase of host-produced ROS (H 2 O 2 ) could be indirect as a result of increased production/secretion of a defense-triggering host response. Moreover, overexpression of TaMIF1 can affect cell morphology, and yeast cells were found to be more sensible to H 2 O 2 , hinting that TaMIF1 may be involved in the regulation of ROS. Thus, we concluded that TaMIF1 might negatively regulate the plant immune system to Pst infection through the repression of ROS accumulation at the infection site, thereby promoting Pst infection. However, the mechanism of TaMIF1 regulating ROS remains to be investigated.

Conclusions
The present study showed that MIFs are important endocrine immune factors in vertebrates. MIF homologs have also been found in all higher plants, but studies on the role of MIFs in plants are limited. In this study, a function of MIFs (TaMIF1) in the wheat innate immune system was uncovered for the first time. It could be speculated that MIF is involved in pathogen-host interaction in both vertebrates and plants. This study demonstrated the functions of TaMIF1 in tautomerase activity and suppressing PCD, which are consistent with MIF in vertebrates. TaMIF1 was shown to be localized in both the cytosol and nuclei in wheat protoplast. In addition, we investigated that TaMIF1 is upregulated in the process of wheat-Pst interaction and TaMIF1 functions as a negative regulator in wheat resistance to Pst dependent on limiting ROS release. The proposed results hinted that TaMIF1 mainly modulates the ROS signaling, and then alters the subsequent immune responses. Nevertheless, the molecular mechanism of TaMIF1 is still unclear and requires further exploration. In the future, our work should focus on seeking the possible targets of TaMIF1 that can help to explore its possible role in wheat immunity. Further, because HsMIF has multiple functions in a variety of biological processes [10], other functions of MIFs in plants deserve further studies. The function characterization of wheat MIF in this study should aid in understanding the biological role of MIFs across kingdoms and be helpful for future in depth functional mechanism analysis on these proteins.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/biology10090878/s1, Figure S1: Sequence alignment of TaMIF coding sequences (cds) on chromasome 5 with two transcripts. Sequences generating identical nucleotide sequences are colored in yellow, those yielding highly similar (>75%) se-quences are shown in blue, and those yielding similar (>50%) residues are shown in green. The primers used for RT-qPCR are indicated in red boxes. Figure S2: Pure proteins analysis through SDS-PAGE. Figure S3: The Western blot of GFP and the TaMIF1-GFP fusion protein with GFP an-tibodies. Figure S4: Western blot analysis of protein expressions in N. benthamiana. Avr1b, TaMIF1 and eGFP were detected with an Flag-antibody. Bax was detected with an HA-antibody. Figure S5: Fungal growth of CYR31 in wheat leaves inoculated with BSMV:00 or BSMV:TaMIF1 at 24 hpi. (A) After staining with wheatgerm agglutinin (WGA), fungal structure was examined under a fluorescence microscope. Bars = 20 µm. (B) Length of Infective hypha was measured us-ing DP-BSW software. The statistical results were obtained from 50 infection sites and values represent mean±SE of three independent replicates. Differences were assessed using Student's t-tests. Asterisks indicate P < 0.05. SV, substomatal vesicle; IH, infection hypha; HMC, haustorial mother cell. Table S1: Primers used in this study. Table S2: RNAi targets predicted of TaMIF1-1as and TaMIF1-2as. The nucleic acid sequences of TaMIF1-1as and TaMIF1-2as were used for predic-tion with the RNAi off-target prediction tool SI-FI 3.2 in both the genome of wheat and Pst. All predicted targets were listed, and they were all from wheat encoding TaMIF1.